Abstract Extensive amounts of research has been done individually on the structure and functional roles ofthe four major glycan types (e.g., N-linked, O-linked, glycosaminoglycan (GAG) andglycosphingolipids (GSL)) in relation to disease states. How these different glycans are actuallydistributed within tissues has barely been studied, and frequently each class is studiedindependent of one another. Furthermore, analysis of these glycan classes are usually done intissue or cell extracts, forfeiting any localization opportunities. Any prior tissue mapping that hasbeen done has relied on broad class carbohydrate binding lectins, or carbohydrate antigenantibodies. These reagents may be used to localize structural glycans motifs within a giventissue, but they do not generally distinguish N-glycan or O-glycan proteins, nor GSL structures,as these could all share the same glycan structural target. To address this, our lab has recentlydeveloped a novel method to profile N-linked glycans directly on tissue using MALDI-imagingmass spectrometry using a high-resolution MALDI-FTICR instrument. This approach is mosteffective using formalin-fixed tissues, offering an unprecedented opportunity to analyze diseaseand normal formalin-fixed tissue blocks stored world-wide. The method is also very effective atidentification of both N-linked glycans and major GSL species in frozen tissues. The goal of thecurrent proposal is to develop related MALDI tissue imaging methods for O- linked mucin typeglycans and chondroitin/heparin GAG classes. This will provide unprecedented capabilities toexamine not only the individual distributions of these four glycan classes in tissues, but alsoallow comparisons of their distributions together in the same tissues. The proposal directlyaddresses the RFA-RM-15-008 goal of developing analytical methods and tools to enable rapidand detailed characterization of the complete glycan representation from a biological source.